Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.     

Studies on the role of eukaryotic nucleotide exchange factor in polypeptide chain initiation

Interactions of eukaryotic 5-dimethylaminonaphthalene-1-sulfonyl-initiation factor 2 (eIF-2) from rabbit reticulocytes and the guanine nucleotide exchange factor ( GEF ), Met-tRNAf, GTP, and GDP were monitored by changes in fluorescence anisotropy and radioactive filtration assays. At 1 mM Mg2+, radioactive filtration assays demonstrate that GEF is necessary for nucleotide exchange. We did not observe a GDP dependence in the association reaction of eIF-2 X GEF for GDP concentrations from 0.01 to 20 microM. This is in disagreement with the model: eIF-2 X GDP + GEF in equilibrium eIF-2 X GEF + GDP. The addition of GTP caused a decrease in fluorescence anisotropy which is interpreted as a dissociation of eIF-2 X GEF . We propose an asymmetrical model of ternary complex (eIF-2 X GTP X Met-tRNAf) formation where 1) GDP does not displace GEF and 2) GTP replaces GEF and presumably GDP. For reticulocyte eIF-2, phosphorylation of the alpha subunit greatly inhibits protein synthesis. This inhibition derives neither from failure of GEF to bind to eIF-2(alpha P) nor from greatly enhanced binding of GEF . The inhibition results from the requirement of very high levels of GTP (100 microM) to dissociate the eIF-2(alpha P) X GEF complex.     

Purification and properties of the guanine nucleotide exchange factor (GEF) from HeLa cells and its role in the initiation of protein synthesis

A guanine nucleotide exchange factor (GEF), catalyzing the exchange of GDP bound to initiation factor eIF-2 for GTP, has been isolated from S3 HeLa cells as the eIF-2 X GEF complex and extensively purified by procedures originally developed for purification of GEF from rabbit reticulocytes. The HeLa cell factor resembles rabbit reticulocyte eIF-2 X GEF in polypeptide composition, catalytic activity, and inactivation by alpha-phosphorylated eIF-2.     

Mechanism of the nucleotide exchange reaction in eukaryotic polypeptide chain initiation. Characterization of the guanine nucleotide exchange factor as a GTP-binding protein

Polypeptide chain initiation in mammalian systems is regulated at the level of the guanine nucleotide exchange factor (GEF). This multisubunit protein catalyzes the exchange of GDP bound to eukaryotic initiation factor 2 (eIF-2) for GTP. Although various models have been proposed for its mode of action, the exact sequence of events involved in nucleotide exchange is still uncertain. We have studied this reaction by three different experimental techniques: (a) membrane filtration assays to measure the release of [3H]GDP from the eIF-2.[3H]GDP binary complex, (b) changes in the steady-state polarization of fluorescamine-GDP during the nucleotide exchange reaction, and (c) sucrose gradient analysis of the total reaction. The results obtained do not support the reaction as written: eIF-2.GDP + GEF in equilibrium eIF-2.GEF + GDP. The addition of GEF alone does not result in the displacement of eIF-2-bound GDP. The release of bound GDP is dependent on the presence of both GTP and GEF, and this argues against the possibility of a substituted enzyme (ping-pong) mechanism for the guanine nucleotide exchange reaction. An important finding of the present study is the observation that GTP binds to GEF. The Kd value of 4 microM for GTP was estimated (a) by the extent of quenching of tryptophan fluorescence of GEF in the presence of GTP and (b) by the binding of [3H]GTP to GEF as measured on nitrocellulose membranes. The GEF-dependent release of eIF-2-bound GDP was studied at several constant concentrations of one substrate (GTP or eIF-2.GDP) while varying the second substrate concentration, and the results were then plotted according to the Lineweaver-Burk method. Taken together, the results of GTP and eIF-2.GDP binding to GEF and the pattern of the double-reciprocal plots strongly suggest that the guanine nucleotide exchange reaction follows a sequential mechanism.     

Protein synthesis in Drosophila melanogaster embryos. Purification and characterization of polypeptide chain-initiation factor 2

Eukaryotic initiation factor 2 (elF-2) was purified from the high-salt wash fraction of Drosophila melanogaster embryos. This factor, with a molecular mass of about 90 kDa, consists of two subunits of 47 kDa and 39 kDa on dodecylsulfate/polyacrylamide gel electrophoresis. The 39-kDa subunit is phosphorylated by the hemin-controlled inhibitor of rabbit reticulocytes in a terminal fragment which can be cleaved by mild treatment with trypsin. Drosophila elF-2 is not a substrate for protein kinases capable of phosphorylating the beta subunit of elF-2 from rabbit reticulocytes. It is also shown that Drosophila elF-2 can form a ternary complex with GTP and Met-tRNAi, which can be efficiently transferred to 40S ribosomes in the presence of AUG and Mg2+. This factor is able to form a binary complex with GDP. Furthermore, purified elF-2 contains about 0.3 mol bound GDP/mol suggesting a high affinity of the factor for this nucleotide. Data supporting the notion that this affinity is increased in the presence of Mg2+, which impairs the GDP/GTP exchange on elF-2, are presented. The properties of Drosophila elF-2 suggest that this factor may be susceptible to regulation by a mechanism like that operating on rabbit reticulocyte elF-2.     

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.     

The effect of Mg2+ and guanine nucleotide exchange factor on the binding of guanine nucleotides to eukaryotic initiation factor 2

A major site of regulation of polypeptide chain initiation is the binding of Met-tRNA to 40 S ribosomal subunits which is mediated by eukaryotic initiation factor 2 (eIF-2). The formation of ternary complex, eIF-2.GTP.Met-tRNA, is potently inhibited by GDP. Measurement of the parameters for guanine nucleotide binding to eIF-2 is critical to understanding the control of protein synthesis by fluctuations in cellular energy levels. We have compared the dissociation constants (Kd) of eIF-2.GDP and eIF-2.GTP and find that GDP has a 400-fold higher affinity for GDP than GTP. The Kd for GDP is almost an order of magnitude less than has been reported previously. The difference between the Kd values for the two nucleotides is the result of a faster rate constant for GTP release, the rate constants for binding being approximately equal. This combination of rate constants and low levels of contaminating GDP in preparations of GTP can explain the apparently unstable nature of eIF-2.GTP observed by others. Mg2+ stabilizes binary complexes slowing the rates of release of nucleotide from both eIF-2.GDP and eIF-2.GTP. The competition between GTP and GDP for binding to eIF-2.guanine nucleotide exchange factor complex has been measured. A 10-fold higher GTP concentration than GDP is required to reduce [32P] GDP binding to eIF-2.guanine nucleotide exchange factor complex by 50%. The relevance of this competition to the regulation of protein synthesis by energy levels is discussed.     

The conversion of eIF-2.GDP to eIF-2.GTP by eIF-2B requires Met-tRNA(fMet).

We have investigated why the recycling of eIF-2.GDP to eIF-2.GTP, mediated by the guanine nucleotide exchange factor eIF-2B, is rapid in rabbit reticulocyte lysate, reconstituted for optimal protein synthesis, but slow in an isolated reaction with purified eIF-2B. We have found that purified eIF-2B dissociates eIF-2.[3H]GDP as efficiently in the presence of GTP as it does in the presence of GDP provided Met-tRNA(fMet) is added. tRNA(fMet) is ineffective, and there is no Met-tRNA(fMet) requirement for exchange with GDP. Exchange of eIF-2 bound GDP for GTP is completely dependent upon Met-tRNA(fMet) in the presence of ATP, suggesting that under physiological conditions efficient recycling of eIF-2.GDP to eIF-2.GTP requires conversion of the latter, a relatively unstable complex, to a more stable Met-tRNA(fMet).eIF-2.GTP complex.     

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.     

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.     

A comparative study of the characteristics of eIF-2 and eIF-2-ancillary factor activities from yeast Saccharomyces cerevisiae and rabbit reticulocytes

The characteristics of yeast eukaryotic initiation factor 2 (eIF-2) and Co-eIF-2A have been studied and compared with those of the corresponding factors from rabbit reticulocytes. 1) Unlike eIF-2r, purified eIF-2y did not contain bound GDP. 2) Purified eIF-2y preparation contained GTPase activity and dephosphorylated GTP to GDP. 3) An anti-eIF-2r preparation which predominantly precipitated the gamma-subunit (Mr 54,000) of eIF-2r also precipitated the larger subunit (Mr 54,000) of eIF-2y. 4) Unlike eIF-2r, ternary complex formation by eIF-2y was not inhibited by Mg2+. 5) Both Co-eIF-2A20y and Co-eIF-2r significantly enhanced Met-tRNAf binding to eIF-2y and, again, Mg2+ did not have any effect on this stimulated Met-tRNAf binding to eIF-2y. 6) Both Co-eIF-2A20y and Co-eIF-2r were similarly effective in stimulating Met-tRNAf binding to eIF-2r in the absence of Mg2+. However, in the presence of Mg2+, Co-eIF-2A20y was significantly less effective than Co-eIF-2r as Co-eIF-2A20y did not promote displacement of GDP from eIF-2r X GDP. 7) eIF-2y bound [3H]GDP and this binding was significantly enhanced in the presence of Mg2+. Also, [3H]GDP in the preformed eIF-2y X [3H]GDP complex was rapidly exchanged with exogenously added unlabeled GDP in the presence of Mg2+. Co-eIF-2A20y had no effect on GDP binding to eIF-2y nor on GDP exchange reactions. 8) Reticulocyte heme-regulated protein synthesis inhibitor, which phosphorylated almost completely (in excess of 80%) the alpha-subunit (Mr 38,000) of eIF-2r, also phosphorylated similarly the smaller subunit (Mr 36,000) of eIF-2y. However, such phosphorylation had no significant effect on ternary complex formation, GDP binding, and GDP exchange reactions.     

Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.     

Mechanism of stimulation of globin synthesis by adenosine-3',5'-monophosphate and guanosine 5'-triphosphate in a rabbit reticulocyte lysate system

The inhibition of globin synthesis in hemin-deficient rabbit reticulocyte lysates is due to the activation of a hemin-controlled translational inhibitor (HCI) that specifically phosphorylates eIF-2 alpha. High concentrations of cAMP (5-10 mM) and GTP (1-2 mM) stimulated the globin synthesis in hemin-deficient lysates when these compounds were added at the initial stage of incubation. The mechanism of the stimulation by cAMP and GTP was studied using hemin-deficient lysates, the N-ethylmaleimide (NEM)-treated HCI-supplemented lysates and a partially purified initiation factor, eIF-2. As the stimulation of globin synthesis by these compounds must be due to the prevention of the inhibition of globin synthesis, or due to the restoration of globin synthesis, or both, the preventive and restorative effects of these compounds were examined. As for the preventive effect, it was observed that a) the activation of HCI in the postribosomal supernatant of reticulocytes was prevented by GTP, but not by cAMP, and b) cAMP and GTP inhibited the phosphorylation of eIF-2 alpha in hemin-deficient lysates. As for the restorative effect of cAMP and GTP, it was observed that c) these compounds restored the globin synthesis and the binding of [35S]Met-tRNAf to the 40S ribosomal subunits, and promoted the dephosphorylation of eIF-2(alpha P), d) the rates of the restored synthesis of globin were lower than the control, and e) cAMP promoted the release of [3H]GDP from the eIF-2(alpha P) X [3H]GDP complex and the formation of eIF-2(alpha P) X eIF-2B complex. Finding (d) indicates that steps involved in the restorative effect of these compounds may not contribute to the stimulation of the globin synthesis in hemin-deficient lysates. The data on the preventive and restorative effects of cAMP and GTP showed that these compounds affected multiple steps. That is, cAMP inhibited the phosphorylation of eIF-2 alpha and promoted both the release of GDP from eIF-2 and the formation of eIF-2(alpha P) X eIF-2B complex, and GTP prevented both the activation of HCI and the phosphorylation of eIF-2 alpha. Though cAMP and GTP affected multiple steps, it is suggested that cAMP stimulates the globin synthesis by inhibiting the phosphorylation of eIF-2 alpha and that GTP stimulates the globin synthesis chiefly by preventing the activation of HCI in hemin-deficient lysates.     

Structural studies on the eukaryotic chain initiation factor 2 from rabbit reticulocytes and brine shrimp Artemia embryos. Phosphorylation by the heme-controlled repressor and casein kinase II

In contrast to reticulocyte polypeptide chain initiation factor 2 (eIF-2), the Artemia factor retains activity in the presence of Mg2+ or after phosphorylation of its alpha-subunit by rabbit reticulocyte heme-controlled repressor (Mehta, H. B., Woodley, C. L., and Wahba, A. J. (1983) J. Biol. Chem. 258, 3438-3441). Furthermore, we have so far been unable to demonstrate a requirement for a GDP/GTP nucleotide exchange factor with Artemia eIF-2. In order to explain these differences we compared the structure of eIF-2 from Artemia and rabbit reticulocytes by using one- and two-dimensional phosphopeptide and iodopeptide maps. Partial trypsin digestion of the alpha-subunit of Artemia eIF-2 after phosphorylation by the heme-controlled repressor generates a 4000 Mr phosphopeptide. Upon extensive trypsin digestion, the two-dimensional phosphopeptide maps of the alpha-subunits for the reticulocyte and Artemia factors are indistinguishable, whereas the iodopeptide maps are different. In addition, immunoblotting indicates that there is no consistent cross-reactivity of the reticulocyte subunits with antibodies prepared in rabbits against the Artemia eIF-2 subunits. A casein kinase II activity was isolated from Artemia embryos that phosphorylates the beta-subunit of reticulocyte eIF-2, but specifically phosphorylates the alpha-subunit of eIF-2 preparations from several non-mammalian sources, including Artemia, yeast, and wheat germ embryos. Since this kinase phosphorylates a site distinct from that recognized by the heme-controlled repressor, and this phosphorylation does not alter the ability of Artemia eIF-2 to undergo nucleotide exchange, caution must be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in non-mammalian cells.     

A GDP/GTP exchange factor essential for eukaryotic initiation factor 2 cycling in Ehrlich ascites tumor cells and its regulation by eukaryotic initiation factor 2 phosphorylation

Formation of the ternary complex Met-tRNAi X eukaryotic initiation factor (eIF) 2 X GTP from eIF-2 X GDP requires exchange of GDP for GTP. However, at physiological Mg2+ concentrations, GDP is released from eIF-2 exceedingly slowly (Clemens, M.J., Pain, V.M., Wong, S.T., and Henshaw, E.C. (1982) Nature (Lond.) 296, 93-95). However, GDP is released rapidly from impure eIF-2 preparations, indicating the presence of a GDP/GTP exchange factor. We have now purified this factor from Ehrlich cells and refer to it as GEF. CM-Sephadex chromatography of ribosomal salt wash separated two peaks of eIF-2 activity. GEF was found in association with eIF-2 in the first peak and co-purified with eIF-2 under low salt conditions. It was separated from eIF-2 in high salt buffers and further purified on hydroxylapatite and phosphocellulose. Gel electrophoresis of our purest preparations showed major bands at 85, 67, 52, 37, 27, and 21 kDa. Purified GEF increased the rate of exchange of [32P] GDP for unlabeled GDP 25-fold but did not function with phosphorylated eIF-2 (alpha subunit). The factor also stimulated markedly the rate of ternary complex formation using eIF-2 X GDP as substrate with GTP and Met-tRNAi but not using phosphorylated eIF-2 X GDP as substrate. eIF-2 is released from the 80 S initiation complex with hydrolysis of GTP. If eIF-2 X GDP is actually the complex released, then GEF is absolutely required for eIF-2 to cycle and it is therefore a new eukaryotic initiation factor. Furthermore, the inability of GEF to utilize eIF-2 (alpha P) X GDP explains how phosphorylation of eIF-2 can inhibit polypeptide chain initiation.     

Purification and characterization of eukaryotic initiation factor 2 and a guanine nucleotide exchange factor from rat liver

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.     

Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.     

Protein synthesis in rabbit reticulocytes. A study of the roles of Co-eIF-2, Co-eIF-2A80, and GDP in peptide chain initiation

The roles of Co-eIF-2, Co-eIF-2A80, and GDP in ternary complex and Met-tRNAf X 40 S initiation complex formation were studied. 1) Partially purified eukaryotic initiation factor 2 (eIF-2) (50% pure) preparations contained 0.4-0.6 pmol of bound GDP/pmol of eIF-2. eIF-2 purity was calculated from ternary complex formation in the absence of Mg2+ and in the presence of excess Co-eIF-2. 2) In the absence of Mg2+, approximately 30% of the potentially active eIF-2 molecules formed ternary complexes, and both Co-eIF-2 and Co-eIF-2A80 were equally effective in full activation of the eIF-2 molecules for ternary complex formation. 3) In the presence of Mg2+, approximately 10% of the potentially active eIF-2 molecules formed ternary complexes in the absence of ancillary factors, and the ancillary factors Co-eIF-2A80 and Co-eIF-2 raised the incorporation to 20 and 50% of the eIF-2 molecules, respectively. 4) In the absence of Mg2+, [3H]GDP in preformed eIF-2 X [3H]GDP was readily displaced by GTP during ternary complex formation. 5) In the presence of Mg2+, [3H]GDP remained tightly bound to eIF-2 and ternary complex formation was inhibited. Co-eIF-2, but not Co-eIF-2A80, was effective in promoting [3H]GDP displacement and the former was more effective in promoting ternary complex formation than the latter. 6) eIF-2 X [3H]GDP was converted to eIF-2 X [3H] GTP by incubation in the presence of nucleoside-5'-diphosphate kinase and ATP, but the eIF-2 X [3H]GTP thus formed did not bind Met-tRNAf in the presence of Mg2+ and required exogeneous addition of Co-eIF-2 and GTP for ternary complex formation and GTP displacement. 7) In the absence of Mg2+, the increased ternary complex formed in the presence of eIF-2 X [3H] GDP and Co-eIF-2A80 (with accompanying loss of [3H] GDP) was inactive in a subsequent reaction, which involves Met-tRNAf transfer to 40 S ribosomes (in the presence of Mg2+), and required trace amounts of Co-eIF-2 for such activity. Based on the above observations, we have suggested a two-step activation of eIF-2 molecules by the Co-eIF-2 protein complex for functional ternary complex formation. One of these steps involves the Co-eIF-2A component of Co-eIF-2. This activation results in stimulated Met-tRNAf binding to eIF-2 and is most apparent in the absence of Mg2+ and with aged eIF-2 molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.